藏药仁青常觉中砷在大鼠尿液中的HPLC-ICP-MS研究
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摘要
目的:采用HPLC-ICP-MS法对藏药仁青常觉中砷(As)在大鼠体内代谢产物—尿液中的形态进行研究。方法:采用Dionex Ion Pac AS 19阴离子色谱柱,以2 mmol·L~(-1)磷酸二氢钠-0.2 mmol·L~(-1)乙二胺四乙酸二钠-10 mmol·L~(-1)无水乙酸钠-3 mmol·L~(-1)硝酸钾(pH=10.5～11.3)-无水乙醇为流动相;流速1.0 mL·min~(-1);进样量10μL,柱温为室温。结果:实验表明,该方法不受~(40)Ar~(35)Cl+干扰,常见的5种As形态化合物的线性范围为0.5～100μg·L-1,甜菜碱(As B)、二甲基胂(DMA)、三价砷[As(Ⅲ)]、一甲基胂(MMA)及五价砷[As(Ⅴ)]的定量下限分别为0.07、0.34、0.135、0.13、0.2μg·L-1,相关系数(R~2)均大于0.999。通过对尿液中As总量及As形态进行分析,结果 As总量在给药第15天达到最大值,主要As形态化合物为As B、DMA、As(Ⅲ)和As(Ⅴ);在第26天即停药后第11天As总量降到最低,主要As形态化合物为As B、DMA和As(Ⅴ)。仁青常觉中As在大鼠体内的代谢研究表明:As(Ⅲ)通过大鼠给药吸收进入体内,后在大鼠体内被部分氧化为As(Ⅴ),经过生物甲基化反应,转化为DMA,最终通过尿液排出体外。4种As形态在大鼠尿液中的排泄速度以DMA最快。结论:藏药仁青常觉中As在大鼠体内代谢产物—尿液中的形态分析的研究,为藏药中As的毒理学研究提供了依据。
Objective:To establish an HPLC-ICP-MS method for the study of the metabolic fate of arsenicspeciation in urine metabolites of Renqing Changjue in rats.Methods:Analysis was performed on a Dionex IonPac AS19 anion chromatographic column,with 2 mmol·L~(-1) dihydrogen phosphate dihydrogen-0.2 mmol·L~(-1) disodium tetraacetic acid disodium-10 mmol·L~(-1) sodium acetate-3 mmol·L~(-1) potassium nitrate(pH=10.5-11.3)-ethyl alcohol as the mobile phase.The flow rate was 1.0 mL·min~(-1),the injection volume was 10 μL,and the column temperature was room temperature.Results:The experiments showed that the method was not affected by ~(40)Ar~(35) Cl~+ interference.The linears of AsB,DMA,As(Ⅲ),MMA and As(Ⅴ)were in the range of 0.5-100 μg·L~(-1) and the limits of quantitation were 0.07,0.34,0.135,0.13 and 0.2 μg·L~(-1),respectively.The correlation coefficients(R~2)were above 0.999.The results of repeatability,accuracy and precision test accorded with the requirements of the measurement.Through the analysis of the content of total arsenic and arsenic speciation in urine samples,the results showed that the total amount of arsenic reached the maximum value on the 15 th day.And the mainly arsenic compounds were presented as AsB,DMA,As(Ⅲ)and As(Ⅴ).On the 26 th day(the 11 th day after discontinuation),urine arsenic reduce to a minimum content.And the major compounds were observed as AsB、DMA and As(Ⅴ).The study of arsenic speciation in vivo rat metabolites indicated that As(Ⅲ)in Renqing Changjue was absorbed into the body,then was partial oxidized to As(Ⅴ),finally was transformed into DMA through biological methylation reaction.Comparing with the other forms of arsenic in rat urine,the DMA had the fastest excretory speed.Conclusion:This study of arsenic speciation in the urine metabolites of rats can provide basis for the research of arsenic toxicology in Tibetan medicine.
Objective:To establish an HPLC-ICP-MS method for the study of the metabolic fate of arsenicspeciation in urine metabolites of Renqing Changjue in rats.Methods:Analysis was performed on a Dionex IonPac AS19 anion chromatographic column,with 2 mmol·L~(-1) dihydrogen phosphate dihydrogen-0.2 mmol·L~(-1) disodium tetraacetic acid disodium-10 mmol·L~(-1) sodium acetate-3 mmol·L~(-1) potassium nitrate(pH=10.5-11.3)-ethyl alcohol as the mobile phase.The flow rate was 1.0 mL·min~(-1),the injection volume was 10 μL,and the column temperature was room temperature.Results:The experiments showed that the method was not affected by ~(40)Ar~(35) Cl~+ interference.The linears of AsB,DMA,As(Ⅲ),MMA and As(Ⅴ)were in the range of 0.5-100 μg·L~(-1) and the limits of quantitation were 0.07,0.34,0.135,0.13 and 0.2 μg·L~(-1),respectively.The correlation coefficients(R~2)were above 0.999.The results of repeatability,accuracy and precision test accorded with the requirements of the measurement.Through the analysis of the content of total arsenic and arsenic speciation in urine samples,the results showed that the total amount of arsenic reached the maximum value on the 15 th day.And the mainly arsenic compounds were presented as AsB,DMA,As(Ⅲ)and As(Ⅴ).On the 26 th day(the 11 th day after discontinuation),urine arsenic reduce to a minimum content.And the major compounds were observed as AsB、DMA and As(Ⅴ).The study of arsenic speciation in vivo rat metabolites indicated that As(Ⅲ)in Renqing Changjue was absorbed into the body,then was partial oxidized to As(Ⅴ),finally was transformed into DMA through biological methylation reaction.Comparing with the other forms of arsenic in rat urine,the DMA had the fastest excretory speed.Conclusion:This study of arsenic speciation in the urine metabolites of rats can provide basis for the research of arsenic toxicology in Tibetan medicine.
引文
[1]热增才旦,张兰,利毛才让,等.微波消解ICP-MS法测定藏药仁青常觉中的无机元素[J].青海师范大学学报(自然科学版),2016(4):59Re-Zeng-Cai-Dan,ZHANG L,Li-Mao-Cai-Rang,et al.Determination of the inorganic elements in Renqing Changjue by ICP-MS[J].J Qinghai Norm Univ(Nat Sci),2016(4):59
[2]魏盛,朱德豪,张克升,等.仁青常觉治疗MNNG致大鼠慢性萎缩性胃炎的实验研究[J].中药新药与临床药理,2015,26(1):52WEI S,ZHU DH,ZHANG KS,et al.Experimental research of Renqing Changjue for treatment of MNNG-induced rat chronic atrophic gastritis[J].Tradit Chin Drug Res Clin Pharmacol,2015,26(1):52
[3]王杰琼,王美艳,薛玲,等.仁青常觉对慢性萎缩性胃炎模型大鼠胃黏膜病理学的影响[J].山东中医药大学学报,2015,35(4):358WANG JQ,WANG MY,XUE L,et al.Effect of Renqing Changjue on pathology of gastric mucosa of rats with chronic atrophic gastritis[J].J Shandong Univ Tradit Chin Med,2015,35(4):358
[4]马文俊,孙雪梅,李宝文,等.藏药仁青常觉的毒理学实验研究[J].亚太传统医药,2013,9(2):15MA WJ,SUN XM,LI BW,et al.Toxicological experimental study of Tibetan medicine Renqing Changjue[J].Asia Pac Tradit Med,2013,9(2):15
[5]房少新,洛桑扎西.藏药仁青常觉中微量元素的测析[J].西北民族大学学报(自然科学版),2012,33(3):63FANG SX,Luo-Sang-Zha-Xi.Measurement of trace element in Renqing Changjue[J].J Northwest Univ Natl(Nat Sci),2012,33(3):63
[6]王英锋,竺梅,冯敏,等.仁青芒觉中可溶性砷形态的HPLC-ICPMS研究[J].药物分析杂志,2011,31(10):1884WANG YF,ZHU M,FENG M,et al.Speciation analysis of solvable arsenic in Renqing Mangjue by HPLC-ICP-MS[J].Chin J Pharm Anal,2011,31(10):1884
[7]陈保卫,那仁满都拉,吕美玲,等.砷的代谢机制、毒性和生物监测[J].化学进展,2009,21(23):474CHEN BW,Na-Ren-Man-Du-La,LüML,et al.Metabolism,toxicity,and biomonitoring of arsenic species[J].Prog Chem,2009,21(23):474
[8]徐文峰,金鹏飞,徐硕,等.电感耦合等离子质谱在药物分析中的应用[J].药物分析杂志,2017,37(12):2123XUWF,JINPF,XUS,etal.ApplicationofICP-MSin pharmaceutical analysis[J].Chin J Pharm Anal,2017,37(12):2123
[9]曹璨,刘成雁,王志嘉,等.微波酸提取/液相色谱-电感耦合等离子体质谱法测定动物源性中药中6种砷形态[J]分析测试学报,2017,36(6):756WANG C,LIU CY,WANG ZJ,et al.Determination of six arsenic species in animal derived traditional Chinese medicines by liquid chromatography combined with inductively coupled plasma mass spectrometry assisted by microwave acid extraction[J].J Instrum Anal,2017,36(6):756
[10]吕翔,王佳佳,邵霞,等.砷元素形态分析和在中药研究中的应用进展[J].中成药,2017,39(8):1679LüX,WANG JJ,SHAO X,et al.Speciation analysis of arsenic and its application progress in traditional Chinese medicine[J].Chin Tradit Pat Med,2017,39(8):1679
[11]陈绍占,杜振霞,刘丽萍,等.高效液相色谱-电感耦合等离子质谱法分析雄黄在大鼠脏器中代谢的砷形态[J].分析化学,2014,42(3):349CHEN SZ,DU ZX,LIU LP,et al.Analysis of arsenic metabolites of realgar in rat viscera by HPLC-ICP-MS[J].Chin J Anal Chem,2014,42(3):349
[12]王博,张浩然,陆淳,等.高效液相色谱-电感耦合等离子体-串联质谱法同时测定饲料中As(Ⅲ)、As(Ⅴ)、氨苯胂酸和洛克沙胂[J].分析科学学报,2018,34(1):100WANG B,ZHANG HR,LU C,et al.Simultaneous determination of A s(Ⅲ),As(Ⅴ),p-ASA and ROX in feeds by high performance liquid chromatography-inductivily coupled plasma tandem mass spectrometry[J].J Anal Sci,2018,34(1):100
[13]林立,陈光,陈玉红.液相色谱-电感藕合等离子体质谱法联用测定动物性海产品中的无机砷[J].环境化学,2009,28(1):149LIN L,CHEN G,CHEN YH.Determination of inorganic arsenic speciation in animal seafood by HPLC-ICP-MS[J].Environ Chem,2009,28(1):149
[14]BRISBIN JA,HYMER CB,CARUSO JA.A gradient anion exchange chromatographic method for the speciation of arsenic in lobster tissue extracts[J].Talanta,2002,58(1):133
[15]RONKART SN,LAURENT V,CARBONNELLE P,et al.Speciation of five arsenic species(arsenite,arsenate,MMAAV,DMAAV and AsBet)in different kinds of water by HPLC-ICP-MS[J].Chemosphere,2007,66(4):738
[16]刘崴,胡俊栋,杨红霞,等.高效液相色谱-电感耦合等离子体质谱法快速测定天然水中无机砷的形态[J].分析试验室,2014,33(4):462LIU W,HU JD,YANG HX,et al.Rapid determination of inorganic arsenic in natural water by high performance liquid chromatographyinductively coupled plasma mass spectrometry[J].Chin J Anal Lab,2014,33(4):462
[17]汤毅珊,王宁生,宓穗卿,等.雄黄中的砷及其代谢物在大鼠体内的排泄动力学研究[J].中药药理与临床,2009,25(3):39TANG YS,WANG NS,MI SQ,et al.Excretion of arsenic in realgar and its metabolites in rats[J].Pharmacol Clin Chin Mater Med,2009,25(3):39
[2]魏盛,朱德豪,张克升,等.仁青常觉治疗MNNG致大鼠慢性萎缩性胃炎的实验研究[J].中药新药与临床药理,2015,26(1):52WEI S,ZHU DH,ZHANG KS,et al.Experimental research of Renqing Changjue for treatment of MNNG-induced rat chronic atrophic gastritis[J].Tradit Chin Drug Res Clin Pharmacol,2015,26(1):52
[3]王杰琼,王美艳,薛玲,等.仁青常觉对慢性萎缩性胃炎模型大鼠胃黏膜病理学的影响[J].山东中医药大学学报,2015,35(4):358WANG JQ,WANG MY,XUE L,et al.Effect of Renqing Changjue on pathology of gastric mucosa of rats with chronic atrophic gastritis[J].J Shandong Univ Tradit Chin Med,2015,35(4):358
[4]马文俊,孙雪梅,李宝文,等.藏药仁青常觉的毒理学实验研究[J].亚太传统医药,2013,9(2):15MA WJ,SUN XM,LI BW,et al.Toxicological experimental study of Tibetan medicine Renqing Changjue[J].Asia Pac Tradit Med,2013,9(2):15
[5]房少新,洛桑扎西.藏药仁青常觉中微量元素的测析[J].西北民族大学学报(自然科学版),2012,33(3):63FANG SX,Luo-Sang-Zha-Xi.Measurement of trace element in Renqing Changjue[J].J Northwest Univ Natl(Nat Sci),2012,33(3):63
[6]王英锋,竺梅,冯敏,等.仁青芒觉中可溶性砷形态的HPLC-ICPMS研究[J].药物分析杂志,2011,31(10):1884WANG YF,ZHU M,FENG M,et al.Speciation analysis of solvable arsenic in Renqing Mangjue by HPLC-ICP-MS[J].Chin J Pharm Anal,2011,31(10):1884
[7]陈保卫,那仁满都拉,吕美玲,等.砷的代谢机制、毒性和生物监测[J].化学进展,2009,21(23):474CHEN BW,Na-Ren-Man-Du-La,LüML,et al.Metabolism,toxicity,and biomonitoring of arsenic species[J].Prog Chem,2009,21(23):474
[8]徐文峰,金鹏飞,徐硕,等.电感耦合等离子质谱在药物分析中的应用[J].药物分析杂志,2017,37(12):2123XUWF,JINPF,XUS,etal.ApplicationofICP-MSin pharmaceutical analysis[J].Chin J Pharm Anal,2017,37(12):2123
[9]曹璨,刘成雁,王志嘉,等.微波酸提取/液相色谱-电感耦合等离子体质谱法测定动物源性中药中6种砷形态[J]分析测试学报,2017,36(6):756WANG C,LIU CY,WANG ZJ,et al.Determination of six arsenic species in animal derived traditional Chinese medicines by liquid chromatography combined with inductively coupled plasma mass spectrometry assisted by microwave acid extraction[J].J Instrum Anal,2017,36(6):756
[10]吕翔,王佳佳,邵霞,等.砷元素形态分析和在中药研究中的应用进展[J].中成药,2017,39(8):1679LüX,WANG JJ,SHAO X,et al.Speciation analysis of arsenic and its application progress in traditional Chinese medicine[J].Chin Tradit Pat Med,2017,39(8):1679
[11]陈绍占,杜振霞,刘丽萍,等.高效液相色谱-电感耦合等离子质谱法分析雄黄在大鼠脏器中代谢的砷形态[J].分析化学,2014,42(3):349CHEN SZ,DU ZX,LIU LP,et al.Analysis of arsenic metabolites of realgar in rat viscera by HPLC-ICP-MS[J].Chin J Anal Chem,2014,42(3):349
[12]王博,张浩然,陆淳,等.高效液相色谱-电感耦合等离子体-串联质谱法同时测定饲料中As(Ⅲ)、As(Ⅴ)、氨苯胂酸和洛克沙胂[J].分析科学学报,2018,34(1):100WANG B,ZHANG HR,LU C,et al.Simultaneous determination of A s(Ⅲ),As(Ⅴ),p-ASA and ROX in feeds by high performance liquid chromatography-inductivily coupled plasma tandem mass spectrometry[J].J Anal Sci,2018,34(1):100
[13]林立,陈光,陈玉红.液相色谱-电感藕合等离子体质谱法联用测定动物性海产品中的无机砷[J].环境化学,2009,28(1):149LIN L,CHEN G,CHEN YH.Determination of inorganic arsenic speciation in animal seafood by HPLC-ICP-MS[J].Environ Chem,2009,28(1):149
[14]BRISBIN JA,HYMER CB,CARUSO JA.A gradient anion exchange chromatographic method for the speciation of arsenic in lobster tissue extracts[J].Talanta,2002,58(1):133
[15]RONKART SN,LAURENT V,CARBONNELLE P,et al.Speciation of five arsenic species(arsenite,arsenate,MMAAV,DMAAV and AsBet)in different kinds of water by HPLC-ICP-MS[J].Chemosphere,2007,66(4):738
[16]刘崴,胡俊栋,杨红霞,等.高效液相色谱-电感耦合等离子体质谱法快速测定天然水中无机砷的形态[J].分析试验室,2014,33(4):462LIU W,HU JD,YANG HX,et al.Rapid determination of inorganic arsenic in natural water by high performance liquid chromatographyinductively coupled plasma mass spectrometry[J].Chin J Anal Lab,2014,33(4):462
[17]汤毅珊,王宁生,宓穗卿,等.雄黄中的砷及其代谢物在大鼠体内的排泄动力学研究[J].中药药理与临床,2009,25(3):39TANG YS,WANG NS,MI SQ,et al.Excretion of arsenic in realgar and its metabolites in rats[J].Pharmacol Clin Chin Mater Med,2009,25(3):39